A Guide to XMRV Research: the WPI Answers Back

February 19, 2010

Posted by Cort Johnson

The inability of the second UK XMRV study – this time from a ‘friendly’ research group headed by Dr. Groom – to find any XMRV in a very large sample of patients was rough news for sure. The ME Action Group in the UK took a rather resigned tone in their response while Dr. Vernon highlighted a few methodological issues but mainly concentrated on questions about that original cohort. Neither presented much good news for CFS patients as a cohort answer to the current problems would mean the virus is only present in a very select subset of patients.

In their response to the latest paper the WPI defined the pitfalls they believe researchers face in validating their work – thus basically giving them a guide on how to find XMRV – and doing everybody a big favor.

No Replications Studies Yet Done: They noted that no one has yet attempted to replicate, i.e. exactly duplicate, their original study. In the best of worlds, of course, a true replication study isn’t necessary and is, in fact, irrelevant. How one found the virus, after all, is not particularly important; pathogens can be uncovered by several techniques and researchers typically use different techniques to validate the presence of a pathogen. In fact, a positive validation study using a different technique is considerably more valuable than a replication study because it definitively demonstrates the pathogen is there. It’s only when the validation studies are unable to validate a finding that the issue of a true replication study becomes important – as it now has.

Looking Back – This replication/validation issue was prominent in Dr. DeFreitas retrovirus of almost 20 years ago. The CDC and Gow teams were well versed in retrovirology and had used standard procedures again and again to find viruses. Given their track record they felt little need to change their procedures. (Ultimately the CDC did at least to some degree). But Dr. DeFreitas felt her bug was different. Given her inability to replicate her results she may have been wrong; the question now is whether XMRV is different as well.

Both UK studies used standard XMRV samples to ensure they could find the virus – and their results indicated that they could – but they couldn’t find it in the CFS patients. It may be important, though, that outside of one Japanese study these are the first attempts to find XMRV in the blood and researchers are treading new ground here.

We know that two US prostate cancer studies found XMRV but two German ones did not. We know the German prostate cancer researcher is redoing his study using a different technique. It’s clear that, irrespective of CFS, the field of XMRV research (as small as it is), is quite muddled at this point – perhaps we shouldn’t be so surprised about the bumps in the road encountered thus far.

The WPI took the UK study to task somewhat for not using their reagents, blood, etc. stating that there is only one way to look for XMRV in the blood that’s been validated and that’s their approach and they have a point. The WPI was the first group to ever look for XMRV in the blood and they validated their results as best they could using the Cleveland Clinic and NCI labs. To be fair the Groom study researchers, some of whom have long track records in CFS research, didn’t have any reason to think their procedures wouldn’t work since apparently they do work for most viruses. It’s possible that both they and the Imperial College researchers underestimated the difficulty of finding this virus in the blood.

To their credit they were careful not to overstate their case simply stating in the paper they were unable to find XMRV DNA in their samples and not making broad conclusions about XMRV and CFS. Once the paper came out they’ve stayed out of the spotlight – - they appear to be waiting to see what other studies turn up.

The WPI’s Issues

Most of the issues pointed out by the WPI don’t appear by themselves to be able to account for the differing results in the UK. String them together, though, and you get an interesting scenario.

Blood Harvesting and Storage – The idea that different blood storage and harvesting procedures could’ve altered the results seems possible but seems unlikely (to this laymen) given that the Groom study used three cohorts from three locations -each of which could have used different storage techniques. We know that XMRV is robust enough for Dr. Peterson to be able to pull XMRV out of a 20 year frozen sample but the possibility does remain that the Groom study inadvertently used blood storage techniques suitable for other viruses but not for XMRV. Laymen’s Conclusion – possibly a significant factor but not likely.

Different Patients - The idea that the WPI had one set of patients and everyone else had a very different group has come up again and again. The very large size of the British study with patients from several different groups appears to make this scenario an unlikely one (and a decidedly unattractive one since then XMRV would apply only to very special patient groups.) On the other hand some differences in patient selection could start to lower the prevalence rate. Laymen’s conclusion – not ‘It’- but a possibly a contributing factor.

Different Geographical Prevalence - that XMRV is simply not found in the UK (but is found in the US and Japan) seems have little plausibility given the fact both Dr. Mikovits and VIP Dx labs have stated they’ve found XMRV in samples from UK patients but it’s certainly possible that XMRV could be less prevalent there thus driving down the prevalence a bit (more?). Laymen’s conclusion – not It either but prevalence rates could be lower – we just don’t know.

Very , Very Low Levels of a Very Difficult to Detect Virus – While the other issues could reduce an investigators ability to find the virus with the exception of the blood storage issue it’s hard to believe they could result in being unable to find any XMRV in a large group of patients. This low viral level issue seems to be more significant, however.

The WPI did two things the other groups didn’t do to find the virus.

They Usually Grew the Virus in White Blood Cells First in Order to Increase its Numbers. (This presumably involves hitting the white blood cells with a substance designed to enhance viral replication). Dr. Mikovits reported earlier that while XMRV appears to be able to readily infect immune cells it simply doesn’t have the tools to replicate readily in them – hence its viral loads are expected to be low. Three sections of the Science paper detail the cultures the WPI used – a clear indication that the viral levels were very low yet the Groom study used uncultured cells. From a laymen’s perspective this is inexplicable. (Did they think the increased sensitivity of their testing made up for that?) One wonders, as they waded through hundreds of negative results if they ever decided to try to culture the cells first! Note, though that the WPI said they usually, but not always, had to use this technique.

WPI researchers Had to Search Multiple Times Both in Time and Space to Find the Virus - Not only did they look at a sample multiple times sometimes they had to look at samples taken at different times from the same patient in order to find the virus. The UK studies, on the other hand, presumably looked at the same samples once or twice. (The Retrovirology study looked at their samples twice, once using a more sensitive technique).

Does perhaps a bit different cohort, perhaps reduced rates of XMRV infection in the UK, maybe some blood storage issues and a much more difficult to find than expected virus equal zero findings in two UK studies? With true replication studies purportedly on the horizon we’ll know in the not too distant future.

A Confident Group – The WPI asserted, as well, that the best test of XMRV infection, given the difficulty finding it using PCR, is an antibodies test. The Retrovirology group did use an antibodies test but the WPI asserted that it was flawed and theirs is superior.

At the end they stated the only reliable to find XMRV in CFS is to use their approach and basically that no-one’s going to find it until they start using their techniques.

I think it was the MEAssociation, not ME ActionUK, who issued a response. I also am not sure about the looking several times part- “Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells“. That sounds like they were able to find it after culturing the samples, it was only if they didn’t culture the samples that they needed to look several times. There’s so many statements that are so hard to tell whether they’re in reference to specific instances or to the techniques in general!

Very nice John – thanks for clarifying that – freshly isolated blood required four passes at different times from a patient – it was remarkably difficult to find this virus. I wonder if this was true for the original Science cohort or if they discovered that later (?). In any case if its that difficult to find the virus it would have helped greatly if they’d been very clear about the measures needed. Perhaps they did state that in the Science paper – I didn’t see them after a quick look through.

If I understood correctly from Dr. Mikovits’ recent public talk, the WPI’s key method was to bring the patient cells in contact with a cell line called “LNCaP”, a strain of rapidly replicating cancer cells. If the virus is present in the patient cells, it will infect the LNCaP cells, which then, thanks to their fast growth, will produce a lot of copies of the virus.

By the way, from a meta point of view, the difficulties with finding XMRV are unsurprising. If it were easy, people would have found it much earlier, perhaps at the same time as HIV. Also, it is easier to find the pathogen in comparatively more virulent diseases like, say, Ebola, simply because they “kill you right away” which means high bacterial/toxin/viral load. It’s no surprise that medicine has discovered the causes for very deadly and disfiguring diseases first.

This was just posted by someone on the Phoenix Rising Forums regarding the efficacy of the Groom Study

“None of the uk patients were diagosed according to the canadian criterea the vast majority of patients supplied were diagnosed according to the oxford criterea.Drawing blood and storing blood for
virological purposes has far more exacting requirements than for routine blood tests as done in England. They would not use the same tubes, anticoagulents, storage temp freezing protocols etc.I’ve attached a post showing the protocol for AIDS testing. Note they do not use whole blood here either but actvated PBMC’s.

During my days as a jobbing microbiologist I was always told that if you are looking for a latent virus you must increase the concentration of your sample and look in the right cells or you are wasting your time and money The WPI techniques were consistent with looking fora virus in its latent phase the British techniques were not—-no blame attached but they didn’t realise what they were dealing this. Their techniques would not have been able to detect latent epstein Barr for example and that is well documented”

I will say that after looking at the methods it’s really unclear to me – a laymen, for sure – why the Groom study didn’t culture their cells. I believe there’s only one reason to culture cells and that’s to up level of a low level virus. The Science paper has three sections where they culture the cells; they obviously did that for a reason – yet the Groom group ignored it and simply used unstimulated cells. It seems inexplicable to me that they could have ignored such a fundamental aspect of the study. Perhaps they thought their increased sensitivity would have made up for that.

Someone on the Phoenix Rising Forums just posted a list of standard blood isolation and amplification procedures for HIV http://forums.aboutmecfs.org/showthread.php?3133-XMRV-CFS-UK-study-II/page52 and stated that the WPI followed most of them and the UK groups followed few of them. If you look at the methods sections of both papers you’ll see that the methods section for the relevant procedures are longer in the WPI paper than in the UK papers.

I’m very surprised that these UK researchers (NOT Wessley and those guys) were not exceedingly careful to take the steps necessary to find the virus (i.e., following the WPI’s testing procedure). Surely they were HIGHLY motivated to find the virus, for two reasons:

1) Not finding it — as we have seen and as could have been easily predicted — has created a backlash against the WPI findings and undoubtedly diminished the interest of many researchers/scientists who were intrigued but skeptical when the WPI findings were originally announced. Not finding the virus is a blow to patients and to anyone who is committed to solving the riddle of ME/CFS, as these researchers surely are.
2) Finding it would have brought to the UK researchers a lot more attention and a LOT more research money than not finding it has.

How scientists this smart, and this committed to conquering ME/CFS, failed to be careful in their methods is beyond baffling. It is morally indefensible.

Cort, it’s time to understand that you aren’t reporting the facts. You’ve, once again, referred to Dr. Elaine DeFreitas’ work yet you seem to be totally unaware that the National CFIDS Foundation had her work replicated by funding well-known researchers in Wisconsin and she helped with that effort. Her discovery had nothing to do with the XMRV virus.

Gee Vickie on a prior post I gave you the link to the NCF’s website which stated that they were unable to find the HTLV-1 in CFS patients. My reference to it was in the form of an analogy; I didn’t intend to suggest that her retrovirus was directly involved.

Honestly, Drew – My best guess is that there’s something we’re just not clear about. They wanted to find the virus; they spent a tremendous amount of money trying to find the virus, why they wouldn’t use the best methods is beyond me. Perhaps they thought they were using the best methods – it sure doesn’t seem like it – I’ve gotta think we’re missing something.

Cort, you wrote, “on a prior post I gave you the link to the NCF’s website which stated that they were unable to find the HTLV-1 in CFS patients” but you fail to understand that they only publish a few of their articles and the work was succesfully replicated (no, not by them but funded by them) and announced at an AACFS medical conference. Your analogy is incorrect.

Despite that fact that every AACFS conference has been reported on in the internet there are no reports of positive HTLV findings at them – which is decidedly odd if what you say is true since until XMRV, Dr. DeFreitas discovery was the most spectacular finding in the history of CFS research. Yet you believe the NCF found the virus, announced it – and everyone ignored it. The researchers then decided to not publish it in a scientific journal – because ??? they didn’t the word to get out? The researchers didn’t want the fame from determining that yes, CFS patients do have a retrovirus? The NCF decided to keep the news quiet (as they were trumpeting every other finding they’ve ever had)? Right…….

I feel like I’m typing in a different language, Cort. The work was replicated and it was announced at an AACFS conference. The work was not done by the NCF. They funded the researchers to do it. And Dr. DeFreitas even was more than helpful to them. The NCF reported on the results in their newsletter. They didn’t announce it at the conferencce. The researcher who participated in the work did, Dr. Knox. Dr. DeFreitas never, never said she found HTLV. She said she found something similar to HTLV. Her actual words were “an HTLV-like virus”. And the replicated work funded by the NCF found she was right but it was only in a small minority and not causal. And, yes, it was in the AACFS book. of abstracts. The NCF does not “trumpet” any news. They were trying to find out what Drs. Grossberg, DeFreitas and Martin had found in the early nineties when all had their funding cut by another group. They succeeded in that endeavor. Why don’t you question those that replicated that particular work (Drs. Knox and Carrigan) or Dr. DeFrietas, herself?

Vickie – this really isn’t making much sense. Yes they found it in about 2% of patients or something like that – which is miniscule – and it wasn’t significantly higher than in the controls. I’m really unsure why you’re continuing with this since a finding like that means its not a significant factor in CFS. Essentially it was a failed project – they were unable to validate Dr. DeFreitas findings of an HTLV-like virus in significant numbers of ME/CFS patients. To put it very bluntly it was a waste of money – no fault of the NCF – many research projects with great ideas simply don’t work out.

No, Cort, you’re wrong again! The research team of Dr. Knox and Dr. Carrigan DID validate Dr. Elaine’s DeFreitas’ work but it was not in significant numbers and was reported on quite factually. It’s on the NCF’s website. But you’ve equated it with the XMRV findings which is not correct any more than the fact that her work was never replicated. Dr. DeFreitas is/was quite brilliant. The real crime is that her funding was cut prematurely as was the fundings of others that were and have been found to be “great ideas” that did “work out.” Read, once again, your original blog.

Oh man, these comments were such a frustrating read. I don’t get why NCF supporters are so hostile to Cort. Vickie, in what way is Cort wrong? He is making total sense to me. Why not give a direct reference to where to find this info that it WAS successfully replicated, instead of attacking him for not knowing? When you say ‘not in significant numbers’ but give no further info, it sounds like you are talking about exactly what Cort is – the study showing it wasn’t found in higher numbers than in the controls – but interpreting it differently. Is this true or is it another study? If another study, where do we find the info (direct link, please) and what exactly were the numbers it was found in? If it was the one Cort is talking about, then he is right in saying it was not successfully replicated. If it is another one, then you are not being clear at all. I don’t understand why you speak so uncivilly to Cort, who is making a great deal of sense and being thorough in explaining his point. The NCF has done some good work, but their supporters or members treating people like they are idiots or bad people for not knowing all their work, or having their own interpretation of it, does them no favors.

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